Transcriptional regulator

ABSTRACT

A database search using the sequence of transcriptional regulator “RING3” having a bromodomain identified an EST sequence that is highly homologous with RING3. Using primers prepared based on the EST sequence, a gene encoding a novel transcriptional regulator having a bromodomain has been successfully isolated from a human testis cDNA library by polymerase chain reactions. The results of analysis of the isolated gene revealed that this gene is expressed strongly in testis cells with a potent proliferative ability. The use of the above transcriptional regulator and its gene enables screening candidate compounds for factors interacting with the transcriptional regulator or drugs controlling the activity of the regulator.

[0001] This application is a continuation-in-part of PCT/JP98/01782,filed Apr. 17, 1998, and claims priority from Japanese Application No.9/116402, filed Apr. 18, 1997.

TECHNICAL FIELD

[0002] The present invention relates to a novel transcriptionalregulator containing a bromodomain and a gene encoding it.

BACKGROUND ART

[0003] The bromodomain is a characteristic motif of amino acids found intranscriptional regulators, and is believed to be involved ininteraction with other proteins, such as other transcriptionalregulators. Proteins having a bromodomain usually contain one or two(Tamkun, J. W. et al., (1992) Cell, 68:561-572, Haynes, S. R. et al.,(1992) Nuc. Acids Res., 20:2603), but sometimes as many as fivebromodomain motifs (Nicolas, R. H. and Goodwin, G. H. (1996), Gene,175(12):233-240). This motif is found in a wide variety of animals. Forexample, it is identified in yeast (Winston, F. et al., (1987),Genetics, 115:649-656; Laurent, B. C. et al., (1991), Proc. Natl. Acad.Sci. USA, 88:2687-2691), Drosophila (Digan, M. E. et al., (1986), Dev.Biol., 114:161-169; Tamkun, J. W. et al., (1992), Cell, 68:561-572), andmammals (Denis, G. V. and Green, M. R. (1996), Genes and Devel.,10:261-271; Yang, X. J. et al., (1996), Nature, 382:319-324).

[0004] All transcriptional regulators containing a bromodomain serve tocontrol signal-dependent transcription in actively proliferating cells(Tamkun, J. W. et al., (1992), Cell, 68:561-572; Haynes, S. R. et al.,(1992), Nuc. Acids Res., 20:2603). Due to this feature, it is suggestedthat cancer may develop if the gene for the protein containing abromodomain is not normally controlled. In fact, several studies haveshown that human transcriptional regulators with a bromodomain RING3,p300/CBP, and PCAF may be involved in oncogenesis.

[0005] RING3 was identified during an extensive analysis of thesequences of human class II major histocompatibility systems (Beck etal., (1992) DNA Seq. 2:203-210). The protein encoded by RING3 ishomologous to D26362, a human gene (Nomura et al., (1994) DNA Res.1:223-229) and fsh, a drosophila gene (Digan et al., (1986), Dev. Biol.,114: 161-169). All three genes encode proteins contain two copies of abromodomain and a PEST sequences. The bromodomain is a motif consistingof 59 to 63 amino acid residues and is considered to be involved inprotein-protein interactions. It is found among the transcriptionalregulator proteins (Tamkun, J. W. et al., (1992) Cell, 68:561-572;Haynes, S. R. et al., (1992) Nuc. Acids Res. 20: 2603). The PESTsequence is a cluster of proline (P), glutamic acid (E), serine (S) andthreonine (T), which characterizes the proteins that undergo rapidproteolysis in the cell.

[0006] The protein encoded by RING3 has a molecular weight of 90 kD andhas serine-threonine activity (Denis and Green, (1996) Genes and Devel.10:261-271). Comparison of the sequences of RING3 and fsh with those ofkinase domains of known serine-threonine kinases revealed that thesub-domains of the kinase motif are conserved, though in no particularorder (most of them are similar to the corresponding sub-domains of aproto-oncogene c-mos). Kinase activity of RING3 is stimulated byinterleukin-1 (IL-1) and forskolin, but not by a certain category ofcytokines (Denis and Green, (1996) Genes and Devel. 10:261-271). A closerelationship between kinase activity and growth phase in chronic andacute lymphocytic leukemia suggests the role RING3 plays in theleukemogenesis regulatory pathway (Denis and Green, (1996) Genes andDevel. 10:261-271). The analysis of the drosophila fsh gene suggestedthe interaction with the trithorax transcriptional regulator, a possibletarget for the putative phosphorylative activity of fsh (Digan et al.,(1986) Dev. Biol. 114:161-169; Mozer and Dawid, (1989) Proc. Natl. Acad.Sci. USA 86:3738-3742). The triathorax gene and its homologue ALL-1 havea C4HC3 zinc finger, a motif commonly found among the genes present atleukemia breakpoints (Aasland et al., (1995) Trends Biochem. Sci. 20:56-59; Saha et al., (1995) Proc. Natl. Acad. Sci. USA 92: 9737-9741).

[0007] In addition to RING3, at least two other bromodomain proteins,p300/CBP and PCAF, are associated with oncogenesis. Although p300protein and CBP protein are encoded by different genes, they areextremely closely related, and therefore, they are often calledp300/CBP. Mutations in CBP are often found in familial and sporadiccancers. Mutations in CBP sometimes result in Rubinstein-Taybi syndrome,which causes patients to develop various malignant tumors (Petrij etal., (1995) Nature 376:348-51). Furthermore, CBP is fused with MOZ att(8;6)(p11;p13) translocation (Borrow et al., (1996) Nature Genet.14:33-41). This fusion protein possibly causes leukemogenesis by itsaberrant acetyltransferase activity (Brownwell and Allis, (1996) Curr.Opin. Genet. Devel. 6:176-184). Mutation in p300 is found in sporadiccolon and gastric cancers (Muraoka et al., (1996) Oncogene12:1565-1569), and. p300 has been suggested to be a gene for atumor-suppressing factor located on chromosome 22q. Another fact thatsuggests the role of p300/CBP in cancer is that it interacts with theknown oncogenes. For example, it is a co-activator of c-Myb (Dai, etal., (1996) Genes and Devel. 10:528-540) and c-Fos (Bannister andKouzarides, (1996) Nature 384:641-643) transcriptional factors, to whichthe E1A protein of Adenovirus bind (Yang et al., (1996) Nature382:319-324). The interaction between E1A and p300/CBP is inhibited byPCAF, a bromodomain protein.

[0008] Like p300/CBP, PCAF also has histone acetyltransferase activity.PCAF, when exogenously expressed, can reduce the proliferationassociated with E1A in cultured cells (Yang et al., (1996) Nature382:319-324). Therefore one of the first mechanisms of the activity ofthe E1A oncogene may be to inhibit the interaction between PCAF andp300.

[0009] Thus, it is thought that aberrant regulation of transcriptionalregulators containing a bromodomain may be closely related to variousdiseases, for example, cancer. Transcriptional regulators containing abromodomain have thus recently received much attention as novel targetsfor treating cancer.

DISCLOSURE OF THE INVENTION

[0010] An objective of the present invention is to provide a noveltranscriptional regulator having a bromodomain and DNA encoding saidtranscriptional regulator. Another objective of the present invention isto provide a vector carrying said DNA, a transformant retaining saidDNA, and a process for producing a recombinant protein by utilizing saidtransformant. A further objective of the present invention is to providea method of screening a compound that binds to said transcriptionalregulator.

[0011] To solve the problems described above, the inventors searched adatabase based on the sequence of RING3 transcriptional regulator with abromodomain and found several EST sequences highly homologous to RING3.Primers were then prepared based on one of the EST sequences, andpolymerase chain reaction was performed in a human testis cDNA libraryusing the primers. As a result, a gene encoding a novel transcriptionalregulator with a bromodomain was successfully isolated. By analyzing theexpression of this gene, the inventors discovered that the gene ishighly expressed in the testis cells. Moreover, the inventors found thata factor interacting with the transcriptional regulator, or a candidatepharmaceutical compound that regulates activity of the transcriptionalregulator, can be screened by utilizing the transcriptional regulatorand the gene encoding it.

[0012] Thus, the present invention relates to novel transcriptionalregulators each having a bromodomain and the genes encoding them, and toa method of screening for a related factor or a candidate compound as amedicament using said proteins or genes, and more specifically relatesto:

[0013] (1) a transcriptional regulator having a bromodomain, whichcomprises the amino acid sequence shown in SEQ ID NO:1, or said sequencewherein one or more amino acids are substituted, deleted, or added;

[0014] (2) a transcriptional regulator having a bromodomain, which isencoded by DNA hybridizing with DNA comprising the nucleotide sequenceshown in SEQ ID NO:2;

[0015] (3) DNA coding for the transcriptional regulator according to (1)or (2);

[0016] (4) a vector comprising the DNA according to (3);

[0017] (5) a transformant expressibly retaining the DNA according to(3);

[0018] (6) a method for producing the transcriptional regulatoraccording to (1) or (2), which comprises culturing the transformantaccording to (5);

[0019] (7) an antibody binding to the transcriptional regulatoraccording to (1) or (2);

[0020] (8) a method of screening a compound having binding activity tothe transcriptional regulator according to (1) or (2), wherein themethod comprises

[0021] (a) contacting a sample with the transcriptional regulatoraccording to (1) or (2) and

[0022] (b) selecting a compound having binding activity to thetranscriptional regulator according to (1) or (2);

[0023] (9) a compound having binding activity to the transcriptionalregulator according to (1) or (2), which can be isolated according tothe method of (8);

[0024] (10) the compound according to (9), which is naturally occurring;and

[0025] (11) DNA specifically hybridizing with DNA comprising thenucleotide sequence shown in SEQ ID NO:2 and having at least 15nucleotides.

[0026] Here, the term “transcriptional regulator(s)” means protein(s)that control gene expression, and “bromodomain” means an amino acidmotif conserved among the transcriptional regulators associated withsignal-dependent transcription, wherein said motif is involved inprotein-protein interaction.

[0027] The present invention relates to a novel transcriptionalregulator having a bromodomain. The amino acid sequence of the proteindesignated TSB contained in the transcriptional regulator of the presentinvention is shown in SEQ ID NO:1, and the nucleotide sequence of thecDNA encoding the protein is shown in SEQ ID NO:2. TSB protein isgenerally known as a region involved in interaction with other factorsincluding transcriptional regulators, and it has a bromodomain(s) (aminoacid positions 49-109 and 292-352 of SEQ ID NO:1), a characteristicmotif of transcriptional regulators involved in cancer (FIG. 1). It isalso highly ex pressed in the testis cells (Example 4). These factssuggest that TSB protein, like other transcriptional regulators havingbromodomains, may be involved in cell proliferative diseases andcancers, particularly in testis cancer. In this connection, bromodomainsare thought to play an important role. Thus, TSB protein, or atranscriptional regulator functionally equivalent thereto, can be usedto prevent and treat cell proliferative diseases and cancers.

[0028] The transcriptional regulators of the present invention can beprepared as recombinant proteins generated using a recombinant genetechnique, or as naturally-occurring proteins. The transcriptionalregulators of the present invention include both recombinant andnaturally-occurring proteins. The recombinant proteins can be preparedusing a method such as incorporating DNA encoding a transcriptionalregulator of the present invention as described below (e.g., DNA havingthe nucleotide sequence shown in SEQ ID NO:2) into a suitable expressionvector, which is then introduced into host cells, and purifying theprotein obtained from the transformant. The naturally occurring proteinscan be prepared using a method such as preparing a column which utilizesan antibody obtained from a small animal immunized with the recombinantprotein prepared as above or its partial peptide, and subjecting theextract from a tissue or cells in which a transcriptional regulator ofthe present invention is overexpressed (e.g., testis) to affinitychromatography using said column.

[0029] The present invention also relates to transcriptional regulatorsfunctionally equivalent to the transcriptional regulators of the presentinvention. A method of introducing mutation into amino acids of aprotein to isolate such a protein is well known to one skilled in theart. Thus, it is well within the art of an ordinarily skilled person toisolate a protein functionally equivalent to the TSB protein having theamino acid sequence shown in SEQ ID NO:1 by appropriately modifying, forexample, substituting amino acids without affecting the function of theprotein using a site-directed mutagenesis system using PCR (GIBCO-BRL,Gaithersburg, Md.), a site-directed mutagenesis using oligonucleotides(Kramer, W. and Fritz, H. J. (1987), Methods in Enzymol., 154:350-367),or the similar methods. Mutation in an amino acid of a protein can alsooccur spontaneously. The transcriptional regulators of the presentinvention include those having the amino acid sequence (SEQ ID NO:1) ofthe TSB potein in which one or more amino acids are substituted,deleted, or added, and functionally equivalent to the TSB protein. Thenumber of mutagenized amino acids is not particularly limited as long asit retains function equivalent to the TSB proiten. It is usually 50amino acids or less, preferably 30 amino acids or less, more preferably10 amino acids or less, and most preferably five amino acids or less.

[0030] As another method of isolating a functionally equivalent proteinutilizing a hybridization technique (Sambrook, J. et al., MolecularCloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is wellknown to one skilled in the art. Based on the DNA sequence encoding theTBS protein (SEQ ID NO:2), or the fragment thereof, a person withordinary skill in the art can isolate DNA highly homologous to said DNAsequences using a hybridization technique (Sambrook, J. et al.,Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press,1989) to obtain a protein functionally equivalent to the TBS protein.The transcriptional regulators of the present invention include thoseencoded by DNA that hybridizes with DNA encoding the TBS protein andfunctionally equivalent to the TBS protein. Source organisms used toisolate a functionally equivalent protein includes, for example, mouse,rat, cattle, monkey, and pig as well as human. The hybridization andwashing conditions for isolating DNA encoding a functionally equivalentprotein are defined as low stringency: 42° C., 2×SSC, 0.1% SDS; moderatestringency: 50° C., 2×SSC, 0.1% SDS; and high stringency: 65° C., 2×SSC,0.1% SDS. The higher the temperature, the more highly homologous DNAwill be obtained. High amino acid homology means usually 40% or more,preferably 60% or more, more preferably 80% or more, or most preferably95% or more. The transcriptional regulator obtained by the hybridizationtechnique preferably contains bromodomain(s). It can also contain aserine/threonine kinase domain which functions to phosphorylate otherproteins, PEST sequence which is a characteristic sequence of theproteins undergoing rapid intracellular proteolysis, and a nucleartransport signal which functions to tranport the protein into nucleus.The presence of the bromodomain in the protein can be identified bysearching the bromodomain motif PROSITE database on DNASIS (HITACHISoftware engineering).

[0031] The present invention also relates to DNA encoding a protein ofthe present invention. The DNA of the present invention includes cDNA,genomic DNA, and chemically synthesized DNA, but is not limited theretoas long as it codes for a protein of the present invention. cDNA can beprepared, for example, by designing a primer based on the nucleic acidsequence shown in SEQ ID NO:2 and performing plaque PCR (see Affara, N.A. et al. (1994), Genomics, 22: 205-210). The genomic DNA can beprepared according to a standard technique using, for example, Qiagengenomic DNA kits (Qiagen, Hilden, Germany). The DNA sequence thusobtained can be determined according to a standard technique using acommercially available dye terminator sequencing kit (AppliedBiosystems) and the like. In addition to applying to the production ofrecombinant proteins as described below, the DNA of the presentinvention may be applied to gene therapy and the like.

[0032] The present invention also relates to a vector into which the DNAof the present invention is inserted. The vector of the presentinvention includes various types of vectors, e.g. for expressing theprotein of the present invention in vivo and for preparing recombinantproteins and appropriately selected depending on the purpose. Vectorsused for expressing the protein of the present invention in vivo (inparticular, for gene therapy) include the adenovirus vector pAdexLcw andthe retrovirus vector pZIPneo. An expression vector is particularlyuseful for producing a protein of the present invention. Although thereis no particular limitation to the expression vectors, the followingvectors are preferred: pREP4 (Qiagen, Hilden, Germany) when E. coli isused; SP-Q01 (Stratagene, La Jolla, Calif.) when yeast is used; andBAC-to-BAC baculovirus expression system (GIBCO-BRL, Gaithersburg, Md.)when insect cells are used. A LacSwitch II expression system(Stratagene; La Jolla, Calif.) is advantageous when mammalian cells,such as CHO, COS, and NIH3T3 cells, are used. The DNA of the presentinvention can be inserted into vectors using a standard method.

[0033] The present invention also relates to a transformant expressiblyretaining the DNA of the present invention. The transformants of thepresent invention include one harboring the above-described vector intowhich the DNA of the present invention is inserted and one having theDNA of the present invention integrated into its genome. The DNA of thepresent invention can be retained in the transformant in any form aslong as the transformant expressibly retains the DNA of the presentinvention. There is no limitation to host cells into which a vector ofthe present invention is introduced. If the cells are used to express aprotein of the present invention for the purpose of ex vivo genetherapy, various cells can be used as target cells suited to diseases.Cells such as E. coli, yeast cells, animal cells, and insect cells canbe used for producing the protein of the present invention. The vectorcan be introduced into the cells by methods such as electroporation andthe calcium phosphate method. Recombinant proteins can be isolated andpurified from the transformants generated for producing the saidproteins according to a standard method.

[0034] The present invention also relates to antibodies that bind to thetranscriptional regulators of the present invention. The antibodies ofthe present invention include, but are not limited to, polyclonal andmonoclonal antibodies. Also included are antisera obtained by immunizingan animal such as a rabbit with a protein of the present invention, anyclass of polyclonal or monoclonal antibodies, humanized antibodiesgenerated by gene recombination, and human antibodies. The antibodies ofthe present invention can be prepared according to the following method.For polyclonal antibodies, antisera can be obtained by immunizing asmall animal, such as a rabbit, with a transcriptional regulator of thepresent invention or a partial peptide thereof, then recovering thefractions that only recognize the transcriptional regulator of thepresent invention through an affinity column coupled with thetranscriptional regulator of the present invention. Immunoglobulin G orM can be prepared by purifying the fractions through a Protein A or Gcolumn. For monoclonal antibodies, a small animal, such as a mouse, isimmunized with a transcriptional regulator of the invention, the spleenis removed from the mouse and homogenized into cells, the cells arefused with myeloma cells from a mouse using a reagent such aspolyethylene glycol, and clones that produce antibodies against thetranscriptional regulator of the invention are selected from theresulting fused cells (hybridoma). The hybridoma obtained is thentransplanted into the abdominal cavity of a mouse, and the ascites arerecovered from the mouse. The obtained monoclonal antibodies can then beprepared by purifying, for example, by ammonium sulfate precipitationthrough a Protein A or G column, by DEAE ion exchanging chromatography,or through an affinity column coupled with the protein of the invention.Besides being used to purify or detect the transcriptional regulators ofthe present invention, the antiobodies of the present invention can beused as a drug for suppressing the functions of the transcriptionalregulator of the present invention. When an antibody is used as a drug,a human or humanized antibody is effective with regard toimmunogenicity. A human or humanized antibody can be prepared accordingto methods well known in the art. For example, a human antibody can beprepared by immunizing a mouse whose immune system is replaced by ahuman system with the transcriptional regulator of the presentinvention. A humanized antibody can be prepared by the CDR graftingmethod in which an antibody gene is cloned from monoclonalantibody-producing cells and its antigenic determinant site istransplanted to an existing human antibody.

[0035] The present invention also relates to a method for screening acompound that binds to transcriptional regulators of the presentinvention. The screening method of the present invention includes stepsof (a) contacting a transcriptional regulator of the present inventionwith a test sample and (b) selecting a compound that has bindingactivity for the transcriptional regulator of the present invention.Test samples used for the screening include, but are not limited to, alibrary of synthetic low molecular weight compounds, a purified protein,an expression product of a gene library, a library of synthetic peptidesa cell extract, and a supernatant of the cell culture. Various methodswell known to one skilled in the art can be used for selecting acompound binding to a transcriptional regulator of the presentinvention.

[0036] A protein that binds to a transcriptional regulator of thepresent invention can be screened by West-western blotting comprisingsteps of generating a cDNA library from the tissues of cells expected toexpress the protein that binds to a transcriptional regulator of thepresent invention (e.g., testis) using a phage vector (λgt11, ZAPII,etc.), allowing the cDNA library to express on the LB-agarose plate,fixing the expressed proteins on a filter, reacting them with thetranscriptional regulator of the present invention purified as abiotin-labeled protein or a fusion protein with GST protein, anddetecting plaques expressing the protein bound to the regulator on thefilter with streptavidin or anti-GST antibody (Skolnik, E. Y., Margolis,B., Mohammadi, M., Lowenstein, E., Fisher, R., Drepps, A., Ullrich, A.and Schlessinger, J. (1991), Cloning of PI3 kinase-associated p85utilizing a novel method for expression/cloning of target proteins forreceptor tyrosine kinases, Cell, 65:83-90). Alternatively, the methodcomprises expressing in yeast cells a transcriptional regulator of thepresent invention which is fused with SFR or GAL4 binding region,constructing a cDNA library in which proteins are expressed in a fusionprotein with the transcription activation site of VP16 or GAL4 from thecells expected to express a protein that binds to the transcriptionalregulator of the present invention, introducing the cDNA library intothe above-described yeast cells, isolating the cDNA derived from thelibrary from the detected positive clones, and introducing andexpressing it in E. coli. (If a protein that binds to thetranscriptional regulator of the present invention is expressed, areporter gene is activated by the binding of the two proteins. Thepositive clones can then be identified.) This method can be performedusing Two-hybrid system (MATCHMAKER Two-Hybrid System, MammalianMATCHMAKER Two-Hybrid Assay Kit, MATCHMAKER One-Hybrid System (all fromClontech); HybriZAP Two-Hybrid Vector System (Stratagene) or inaccordance with Dalton, S. and Treisman R. (1992), Characterization ofSAP-1, a protein recruited by serum response factor to the c-fos serumresponse element, Cell, 68:597-612). Another method is to apply aculture supernatant or a cell extract from the cells suspected toexpress a protein which binds to the transcriptional regulator of thepresent invention onto an affinity column coupled with thetranscriptional regulator of the present invention, and purify theprotein specifically bound to the column.

[0037] Also well known to one skilled in the art are a method ofscreening molecules that bind to a transcriptional regulator of thepresent invention by reacting the immobilized transcriptional regulatorof present invention with a synthetic compound, natural substance bank,or a random phage peptide display library, and a method of isolating lowmolecular weight compounds, proteins (or their genes), or peptides whichbind to a transcriptional regulator of the present invention byutilizing the high-throughput technique of combinatorial chemistry(Wrighton, N. C., Farrell, F. X., Chang, R., Kashuyap, A. K., Barbone,F. P., Mulcahy, L. S., Johnson, D. L., Barrett, R. W., Jolliffe, L. K.,Dower, W. J., Small peptides as potent mimetics of the protein hormoneerythropoietin, Science (UNITED STATES) Jul. 26, 1996, 273:458-464;Verdine, G. L., The combinatorial chemistry of nature, Nature (ENGLAND),Nov. 7, 1996, 384:11-13; Hogan, J. C. Jr., Directed combinatorialchemistry, Nature (ENGLAND), Nov. 7, 1996, 384:17-19). The compoundsthus isolated by the screening method of the present invention arecandidates for drugs for enhancing or surpressing the activity of atranscriptional regulator of the present invention. When the compoundsisolated by the screening method of the present invention are used aspharmaceuticals, they can be formulated by a known pharmacologicalprocess. For example, they can be administered to a patient withpharmaceutically acceptable carriers and vehicles (e.g., physiologicalsaline, vegetable oil, a dispersant, a surfactant, and a stabilizer).The compounds may be percutaneously, intranasally, transbronchially,intramuscularly, intravenously, or orally administered, depending ontheir properties.

[0038] The present invention also relates to DNA specificallyhybridizing with DNA coding the TBS protein and having at least 15nucleotides. As used herein, “specifically hybridizing” means that nocross-hybridization occurs between DNA encoding other proteins underconditions of moderate stringency. Such DNA may be used as a probe fordetecting and isolating the DNA encoding the TBS protein, and as aprimer for amplifying the DNA encoding the protein of the presentinvention. Specific examples of the primers include those shown in SEQID NOs:3 and 4.

[0039] An “isolated nucleic acid” is a nucleic acid the structure ofwhich is not identical to that of any naturally occurring nucleic acidor to that of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in mixtures of different (i) DNA molecules, (ii)transfected cells, and (iii) cell clones: e.g., as these occur in a DNAlibrary such as a cDNA or genomic DNA library.

[0040] The term “substantially pure” as used herein in reference to agiven polypeptide means that the polypeptide is substantially free fromother biological compounds, such as those in cellular material, viralmaterial, or culture medium, with which the polypeptide was associated(e.g., in the course of production by recombinant DNA techniques orbefore purification from a natural biological source). The substantiallypure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%)pure by dry weight. Purity can be measured by any appropriate standardmethod, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

[0041] A “conservative amino acid substitution” is one in which an aminoacid residue is replaced with another residue having a chemicallysimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

[0042] As used herein, “percent identity” of two amino acid sequences orof two nucleic acids is determined using the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as inKarlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993).Such an algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleicacid molecules of the invention. BLAST protein searches are performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to a reference polypeptide. To obtain gappedalignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) are used. Seehttp://www.ncbi.nlm.nih.gov.

[0043] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. In case of conflict,the present application, including definitions, will control. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. The materials, methods,and examples are illustrative only and not intended to be limiting.Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044]FIG. 1 shows the nucleic acid sequence of TSB aligned with theamino acid sequence of the open reading frame. The three motifsidentified by the search of the PROSITE database are underlined. Twobromodomains (amino acid positions 49-109 and 292-352) and a PESTsequence (amino acid positions 636-672) are identified.

[0045]FIG. 2 compares the amino acid sequences of the predicted kinasedomains of TSB, RING3, and fsh. The sub-domains of kinase are disclosedin Denis and Green, (1996) Genes and Devel. 10:261-271, and sub-domainsI-II are excluded. The numerals correspond to the translation positionof TSB. The conserved residues are shaded.

[0046]FIG. 3 shows the map location of TSB. The position is indicatedrelative to the positions of the adjacent markers on chromosome 1pdetermined by radiation hybrid analysis.

[0047]FIG. 4A shows the results of Northern blot analysis of TSB in thenormal tissues (Lane 1, heart; Lane 2, brain; Lane 3, placenta; Lane 4,lung; Lane 5, liver; Lane 6, skeletal muscle; Lane 7, kidney; Lane 8,pancreas; Lane 9, spleen; Lane 10, thymus; Lane 11, prostate; Lane 12,testis; Lane 13, ovary; Lane 14, small intestine; Lane 15, colon (mucouslining); and Lane 16, peripheral leukocytes).

[0048]FIG. 4B shows the results of Northern blot analysis of TSB incarcinoma cell lines (Lane 1, promyelocytic leukemia HL-60; Lane 2, HeLaS3 cells; Lane 3, chronic myelocytic leukemia K-56; Lane 4,lymphoblastic leukemia MOLT-4; Lane 5, Burkitt's lymphoma Raji; Lane 6,large intestinal adenocarcinoma SW480; Lane 7, lung carcinoma S549; andLane 8, melanoma G361). The positions of molecular weight markers areindicated on the right.

BEST MODE FOR IMPLEMENTING THE INVENTION

[0049] The following examples illustrate the present invention in moredetail, but are not to be construed to limit the scope of the presentinvention.

EXAMPLE 1 Identification of EST Homologous to RING3 and Isolation of itsFull-Length Sequence

[0050] A BLAST search of the EST database identified numerous ESTshomologous to the DNA sequence of the RING3 gene (Beck et al., (1992),DNA Seq. 2:203-210) used as a probe. Among these ESTs, EST H64204, whichwas derived from a testis-specific cDNA library (Diatchenko et al.,(1996) Proc. Natl. Acad. Sci. USA 93:6025-6030), had 65% sequencehomology to RING3 spanning 285 bp.

[0051] To clone the full-length sequence of EST H64204, PCR primers U(SEQ ID NO:2: AATGTCTCTGCCAAGTCGACAA) and L (SEQ ID NO:4:AGCATCCACAGGACGTTGAAAG) were designed to perform PCR using testis cDNAsas templates. PCR was carried out at 94° C. for 8 min. followed by 35cycles of 94° C. for 30 sec (heat annealing), 60° C. for 1 min(annealing), and 72° C. for 1 min (extension). AmpliTaq gold (PerkinElmer) was used as an enzyme for PCR. This resulted in a PCR product of175 bp. Subsequently, a testis cDNA library was screened (Clontech;HL3024a) using this PCR product as a probe. All the probes were labeledwith [α-³² P]dCTP by random priming and were purified through Chromaspin(10 columns; Clontech). Hybridization was performed in ExpressHybhybridization solution (Clontech) for 1 hour at 65°. The filter waswashed at the final stringency of 1×SSC, 0.1% SDS, 65° C. The sequenceof a cDNA clone that was aligned with the EST sequence was used tore-screen the library. This process was repeated until a series ofoverlapped clones giving a full-length sequence of the coding region ofthe gene was obtained. As a result, a continuous sequence of 3,104 bpencoding 947 amino acids was found in an open reading frame (ORF) at thenucleotide positions 106-2946. The ORF is followed by a short stretch ofa 3′ untranslated region of 60 bp, which is terminated with a poly(A)tail that has a polyadenylated signal (ATTAAA) in the 20 bp upstream ofit. The inventors designated the isolated clone “TSB (testis specificbromodomain). SEQ ID NO:2 shows the nucleotide sequence of TSB alongwith the predicted amino acid sequence thereof. The predicted amino acidsequence is also shown in SEQ ID NO:1. The nucleotide sequences weredetermined with the automated sequencer ABI377 (Perkin Elmer) by use ofABI dye terminator chemistry.

EXAMPLE 2 Homology and Motifs

[0052] RING3 (66% homology, 59% identity spanning 649 amino acids),D26362 (62% homology, 56% identity spanning 649 amino acids), and fsh(62% homology, 56% identity spanning 565 amino acids) were identified asthe amino acid sequences having the highest homology with TBS bysearching protein databases.

[0053] Two bromodomains (amino acid positions 49-109 and 292-352) wereidentified by searching the PROSITE database for the motif of amino acidsequence. A PEST sequence (Rodgers et al., (1986) Science 234:364) wasalso present in amino acid positions 636-672. FIG. 1 shows the locationsof these motifs.

[0054] Since RING3 is also known to have serine-threonine kinaseactivity (Denis and Green, (1996) Genes and Devel. 10:261-271), theamino acid sequence of TSB was compared with that of the predictedkinase domain of RING3 using Bestfit at GCG. The result showed that thepredicted kinase domain of RING3 is very well conserved in TSB (FIG. 2).However, it was found that TBS did not contain sub-domain I, which codesfor the predicted ATP-binding domain, and sub-domain II, which codes forcatalytic lysine, suggesting that the kinase activity of TSB waspossibly lost.

[0055] In addition, since the RING3 protein is known to be localized inthe nucleus, the predicted nuclear transport signal of TSB wasidentified using the PSORT program. As a result, four copies of thenuclear transport signal (in the 488, 489, 575 and 919 positions), eachcopy consisting of four residues, and two copies of Robins and Dingwallconsensus sequence (Robins and Dingwall, (1991) Cell 64: 615-23) (in the445 and 603 positions) were discovered. Thus, like RING3, nuclearlocalization of TSB was also indicated.

EXAMPLE 3 Mapping of TSB

[0056] To identify the locus of TSB, DNA from 24 human/rodent singlechromosomal somatic cell lines obtained from Coriell Cell Repositories,New Jersey (Dubois and Naylor, (1993) Genomics 16:315-319) wereamplified using primers U (SEQ ID NO:3) and L (SEQ ID NO:4).

[0057] A panel of single chromosomal hybrid cell lines was screened forthe TSB-localized region, using primers U (SEQ ID NO:3) and L (SEQ IDNO:4). As a result, the predicted product of 175 bp was amplified onlyin the cell line GM 13139, a single chromosomal cell line for humanchromosome 1. The same primers were used for PCR for a GeneBridge4radiation hybrid panel (Walter et al., (1994) Nature Genetics 7:22-28).The binary codes generated by assessing each hybrid as positive ornegative for the amplification were compared with the analogous codesfor the markers constituting the framework map, using the server locatedat http://www-genome.wi.mit.edu./cgobin/contig/rhmapper.pl. Thisprocedure was repeated to give independent scores. The two experimentsyielded identical binary codes, and TSB was shown to be located onchromosome 1p between markers WI-7719 and WI-3099 (D1S2154) (FIG. 3).

EXAMPLE 4 Analysis of TSB Expression

[0058] Northern analysis of 16 normal tissues was conducted using theprobe prepared by PCR amplification of the testis cDNAs using primers U(SEQ ID NO:3) and L (SEQ ID NO:4). The probe strongly hybridized withmRNA of 3.5 kb and weakly hybridized with that of 4.0 kb (FIG. 4A). Thisresult was consistent with the source testis-specific library of the ESTused to identify TSB (Diatchenko et al., (1996) Proc. Natl. Acad. Sci.USA 93:6025-6030). In addition, the probe cross-hybridized with the twospecies of mRNA (about 2.0 and 4.5 kb) commonly expressed in thesetissues. Since this probe contained the sequence encoding a bromodomain,the transcripts of the two species potentially represent otherbromodomain genes. Furthermore, a panel of mRNA derived from eight tumorcell lines was screened along with the panel of the normal tissues,since, several other testis-specific genes, in particular the MAGEfamily (van der Bruggen et al., (1991) Science 254:1643-1647) areexpressed in tumor tissues. However, expression of TSB was not detectedin any of the cell lines tested (FIG. 4B). Likewise, a panel of 10samples of lung cancer was negative for the TSB expression (data are notshown). The conditions for hybridization were the same as thosedescribed in Example 1.

INDUSTRIAL APPLICABILITY

[0059] The present invention provides a novel transcriptional regulatorhaving a bromodomain, DNA encoding said transcriptional regulator, avector carrying said DNA, a transformant expressibly retaining said DNA,an antibody binding to said transcriptional regulator, and a method ofscreening a compound that binds to said transcriptional regulator. Thetranscriptional regulator of the present invention is considered to forma family together with a transcriptional regulator RING3 that is thoughtto be associated with cancer. It is abundantly expressed in the testis.Accordingly, the transcriptional regulator of the present invention andDNA encoding said transcriptional regulator can be used to screen fortherapeutics to treat diseases such as cell-proliferative diseases andcancer, particularly testis cancer; diseases attributed to aplasia anddysfunction of sperm; or for contraceptives. Antibodies and othercompounds that bind to the transcriptional regulator of the presentinvention can also be used as therapeutics.

1 28 1 947 PRT Homo sapiens 1 Met Ser Leu Pro Ser Arg Gln Thr Ala IleIle Val Asn Pro Pro Pro 1 5 10 15 Pro Glu Tyr Ile Asn Thr Lys Lys AsnGly Arg Leu Thr Asn Gln Leu 20 25 30 Gln Tyr Leu Gln Lys Val Val Leu LysAsp Leu Trp Lys His Ser Phe 35 40 45 Ser Trp Pro Phe Gln Arg Pro Val AspAla Val Lys Leu Lys Leu Pro 50 55 60 Asp Tyr Tyr Thr Ile Ile Lys Asn ProMet Asp Leu Asn Thr Ile Lys 65 70 75 80 Lys Arg Leu Glu Asn Lys Tyr TyrAla Lys Ala Ser Glu Cys Ile Glu 85 90 95 Asp Phe Asn Thr Met Phe Ser AsnCys Tyr Leu Tyr Asn Lys Pro Gly 100 105 110 Asp Asp Ile Val Leu Met AlaGln Ala Leu Glu Lys Leu Phe Met Gln 115 120 125 Lys Leu Ser Gln Met ProGln Glu Glu Gln Val Val Gly Val Lys Glu 130 135 140 Arg Ile Lys Lys GlyThr Gln Gln Asn Ile Ala Val Ser Ser Ala Lys 145 150 155 160 Glu Lys SerSer Pro Ser Ala Thr Glu Lys Val Phe Lys Gln Gln Glu 165 170 175 Ile ProSer Val Phe Pro Lys Thr Ser Ile Ser Pro Leu Asn Val Val 180 185 190 GlnGly Ala Ser Val Asn Ser Ser Ser Gln Thr Ala Ala Gln Val Thr 195 200 205Lys Gly Val Lys Arg Lys Ala Asp Thr Thr Thr Pro Ala Thr Ser Ala 210 215220 Val Lys Ala Ser Ser Glu Phe Ser Pro Thr Phe Thr Glu Lys Ser Val 225230 235 240 Ala Leu Pro Pro Ile Lys Glu Asn Met Pro Lys Asn Val Leu ProAsp 245 250 255 Ser Gln Gln Gln Tyr Asn Val Val Glu Thr Val Lys Val ThrGlu Gln 260 265 270 Leu Arg His Cys Ser Glu Ile Leu Lys Glu Met Leu AlaLys Lys His 275 280 285 Phe Ser Tyr Ala Trp Pro Phe Tyr Asn Pro Val AspVal Asn Ala Leu 290 295 300 Gly Leu His Asn Tyr Tyr Asp Val Val Lys AsnPro Met Asp Leu Gly 305 310 315 320 Thr Ile Lys Glu Lys Met Asp Asn GlnGlu Tyr Lys Asp Ala Tyr Ser 325 330 335 Phe Ala Ala Asp Val Arg Leu MetPhe Met Asn Cys Tyr Lys Tyr Asn 340 345 350 Pro Pro Asp His Glu Val ValThr Met Ala Arg Met Leu Gln Asp Val 355 360 365 Phe Glu Thr His Phe SerLys Ile Pro Ile Glu Pro Val Glu Ser Met 370 375 380 Pro Leu Cys Tyr IleLys Thr Asp Ile Thr Glu Thr Thr Gly Arg Glu 385 390 395 400 Asn Thr AsnGlu Ala Ser Ser Glu Gly Asn Ser Ser Asp Asp Ser Glu 405 410 415 Asp GluArg Val Lys Arg Leu Ala Lys Leu Gln Glu Gln Leu Lys Ala 420 425 430 ValHis Gln Gln Leu Gln Val Leu Ser Gln Val Pro Phe Arg Lys Leu 435 440 445Asn Lys Lys Lys Glu Lys Ser Lys Lys Glu Lys Lys Lys Glu Lys Val 450 455460 Asn Asn Ser Asn Glu Asn Pro Arg Lys Met Cys Glu Gln Met Arg Leu 465470 475 480 Lys Glu Lys Ser Lys Arg Asn Gln Pro Lys Lys Arg Lys Gln GlnPhe 485 490 495 Ile Gly Leu Lys Ser Glu Asp Glu Asp Asn Ala Lys Pro MetAsn Tyr 500 505 510 Asp Glu Lys Arg Gln Leu Ser Leu Asn Ile Asn Lys LeuPro Gly Asp 515 520 525 Lys Leu Gly Arg Val Val His Ile Ile Gln Ser ArgGlu Pro Ser Leu 530 535 540 Ser Asn Ser Asn Pro Asp Glu Ile Glu Ile AspPhe Glu Thr Leu Lys 545 550 555 560 Ala Ser Thr Leu Arg Glu Leu Glu LysTyr Val Ser Ala Cys Leu Arg 565 570 575 Lys Arg Pro Leu Lys Pro Pro AlaLys Lys Ile Met Met Ser Lys Glu 580 585 590 Glu Leu His Ser Gln Lys LysGln Glu Leu Glu Lys Arg Leu Leu Asp 595 600 605 Val Asn Asn Gln Leu AsnSer Arg Lys Arg Gln Thr Lys Ser Asp Lys 610 615 620 Thr Gln Pro Ser LysAla Val Glu Asn Val Ser Arg Leu Ser Glu Ser 625 630 635 640 Ser Ser SerSer Ser Ser Ser Ser Glu Ser Glu Ser Ser Ser Ser Asp 645 650 655 Leu SerSer Ser Asp Ser Ser Asp Ser Glu Ser Glu Met Phe Pro Lys 660 665 670 PheThr Glu Val Lys Pro Asn Asp Ser Pro Ser Lys Glu His Val Lys 675 680 685Lys Met Lys Asn Glu Cys Ile Leu Pro Glu Gly Arg Thr Gly Val Thr 690 695700 Gln Ile Gly Tyr Cys Val Gln Asp Thr Thr Ser Ala Asn Thr Thr Leu 705710 715 720 Val His Gln Thr Thr Pro Ser His Val Met Pro Pro Asn His HisGln 725 730 735 Leu Ala Phe Asn Tyr Gln Glu Leu Glu His Leu Gln Thr ValLys Asn 740 745 750 Ile Ser Pro Leu Gln Ile Leu Pro Pro Ser Gly Asp SerGlu Gln Leu 755 760 765 Ser Asn Gly Ile Thr Val Met His Pro Ser Gly AspSer Asp Thr Thr 770 775 780 Met Leu Glu Ser Glu Cys Gln Ala Pro Val GlnLys Asp Ile Lys Ile 785 790 795 800 Lys Asn Ala Asp Ser Trp Lys Ser LeuGly Lys Pro Val Lys Pro Ser 805 810 815 Gly Val Met Lys Ser Ser Asp GluLeu Phe Asn Gln Phe Arg Lys Ala 820 825 830 Ala Ile Glu Lys Glu Val LysAla Arg Thr Gln Glu Leu Ile Arg Lys 835 840 845 His Leu Glu Gln Asn ThrLys Glu Leu Lys Ala Ser Gln Glu Asn Gln 850 855 860 Arg Asp Leu Gly AsnGly Leu Thr Val Glu Ser Phe Ser Asn Lys Ile 865 870 875 880 Gln Asn LysCys Ser Gly Glu Glu Gln Lys Glu His Pro Gln Ser Ser 885 890 895 Glu AlaGln Asp Lys Ser Lys Leu Trp Leu Leu Lys Asp Arg Asp Leu 900 905 910 AlaArg Pro Lys Glu Gln Glu Arg Arg Arg Arg Glu Ala Met Val Gly 915 920 925Thr Ile Asp Met Thr Leu Gln Ser Asp Ile Met Thr Met Phe Glu Asn 930 935940 Asn Phe Asp 945 2 3104 DNA Homo sapiens CDS (106)...(2946) 2ggcaagatgt tcctgggagg tcaagttaag agtcaaaaat aattcattag atttaacaat 60ttagcatgga catgtacttg tagacaggat tcaaagcagt taaga atg tct ctg cca 117Met Ser Leu Pro 1 agt cga caa aca gct att att gtt aac cct cct cca ccagaa tat ata 165 Ser Arg Gln Thr Ala Ile Ile Val Asn Pro Pro Pro Pro GluTyr Ile 5 10 15 20 aat act aag aaa aat ggg cga ttg aca aat caa ctt cagtat cta caa 213 Asn Thr Lys Lys Asn Gly Arg Leu Thr Asn Gln Leu Gln TyrLeu Gln 25 30 35 aaa gtt gtc cta aag gat tta tgg aag cat agt ttt tca tggccc ttt 261 Lys Val Val Leu Lys Asp Leu Trp Lys His Ser Phe Ser Trp ProPhe 40 45 50 caa cgt cct gtg gat gct gtg aaa cta aag ttg cct gat tat tatacc 309 Gln Arg Pro Val Asp Ala Val Lys Leu Lys Leu Pro Asp Tyr Tyr Thr55 60 65 att ata aaa aac cca atg gat tta aat aca att aag aag cgc ttg gag357 Ile Ile Lys Asn Pro Met Asp Leu Asn Thr Ile Lys Lys Arg Leu Glu 7075 80 aat aaa tat tat gcg aag gct tca gaa tgt ata gaa gac ttc aat aca405 Asn Lys Tyr Tyr Ala Lys Ala Ser Glu Cys Ile Glu Asp Phe Asn Thr 8590 95 100 atg ttc tca aat tgt tat tta tat aac aag cct gga gat gac attgtt 453 Met Phe Ser Asn Cys Tyr Leu Tyr Asn Lys Pro Gly Asp Asp Ile Val105 110 115 ctt atg gca caa gct cta gag aag ctg ttt atg cag aaa tta tctcag 501 Leu Met Ala Gln Ala Leu Glu Lys Leu Phe Met Gln Lys Leu Ser Gln120 125 130 atg cca caa gaa gag caa gtt gtg ggt gtt aag gaa aga atc aagaaa 549 Met Pro Gln Glu Glu Gln Val Val Gly Val Lys Glu Arg Ile Lys Lys135 140 145 ggc act caa cag aat ata gct gtt tct tct gct aaa gaa aaa tcatca 597 Gly Thr Gln Gln Asn Ile Ala Val Ser Ser Ala Lys Glu Lys Ser Ser150 155 160 ccc agc gca aca gaa aaa gta ttt aag cag caa gaa att cct tctgta 645 Pro Ser Ala Thr Glu Lys Val Phe Lys Gln Gln Glu Ile Pro Ser Val165 170 175 180 ttt cct aag aca tct att tct ccc ttg aac gtg gta cag ggagct tca 693 Phe Pro Lys Thr Ser Ile Ser Pro Leu Asn Val Val Gln Gly AlaSer 185 190 195 gtc aac tcc agt tca caa act gcg gcc caa gtt aca aaa ggtgtg aag 741 Val Asn Ser Ser Ser Gln Thr Ala Ala Gln Val Thr Lys Gly ValLys 200 205 210 agg aaa gca gat aca aca act cct gca act tca gca gtt aaagca agt 789 Arg Lys Ala Asp Thr Thr Thr Pro Ala Thr Ser Ala Val Lys AlaSer 215 220 225 agt gaa ttt tct cca aca ttc aca gaa aaa tca gtg gca ctgcca cct 837 Ser Glu Phe Ser Pro Thr Phe Thr Glu Lys Ser Val Ala Leu ProPro 230 235 240 ata aaa gaa aat atg cca aag aat gtt ttg cca gat tct cagcaa caa 885 Ile Lys Glu Asn Met Pro Lys Asn Val Leu Pro Asp Ser Gln GlnGln 245 250 255 260 tat aat gtt gtg gag act gtt aaa gta act gaa caa ttaagg cac tgt 933 Tyr Asn Val Val Glu Thr Val Lys Val Thr Glu Gln Leu ArgHis Cys 265 270 275 agt gag att ctt aaa gaa atg ctt gca aag aaa cat ttttca tat gca 981 Ser Glu Ile Leu Lys Glu Met Leu Ala Lys Lys His Phe SerTyr Ala 280 285 290 tgg ccc ttt tat aat cct gtt gac gtt aat gct ttg ggactc cat aac 1029 Trp Pro Phe Tyr Asn Pro Val Asp Val Asn Ala Leu Gly LeuHis Asn 295 300 305 tac tat gac gtt gtc aaa aat ccg atg gat ctt gga actatt aag gag 1077 Tyr Tyr Asp Val Val Lys Asn Pro Met Asp Leu Gly Thr IleLys Glu 310 315 320 aaa atg gat aac caa gaa tat aag gat gca tac tca tttgcg gca gat 1125 Lys Met Asp Asn Gln Glu Tyr Lys Asp Ala Tyr Ser Phe AlaAla Asp 325 330 335 340 gtt aga tta atg ttc atg aat tgc tac aag tac aatcct cca gat cac 1173 Val Arg Leu Met Phe Met Asn Cys Tyr Lys Tyr Asn ProPro Asp His 345 350 355 gaa gtt gtg aca atg gca aga atg ctt cag gat gttttc gaa acg cat 1221 Glu Val Val Thr Met Ala Arg Met Leu Gln Asp Val PheGlu Thr His 360 365 370 ttt tca aag atc ccg att gaa cct gtt gag agt atgcct tta tgt tac 1269 Phe Ser Lys Ile Pro Ile Glu Pro Val Glu Ser Met ProLeu Cys Tyr 375 380 385 atc aaa aca gat atc aca gaa acc act ggt aga gagaac act aat gaa 1317 Ile Lys Thr Asp Ile Thr Glu Thr Thr Gly Arg Glu AsnThr Asn Glu 390 395 400 gcc tcc tct gaa ggg aac tct tct gat gat tct gaagat gag cga gtt 1365 Ala Ser Ser Glu Gly Asn Ser Ser Asp Asp Ser Glu AspGlu Arg Val 405 410 415 420 aag cgt ctt gca aag ctt cag gag cag ctt aaagct gta cat caa cag 1413 Lys Arg Leu Ala Lys Leu Gln Glu Gln Leu Lys AlaVal His Gln Gln 425 430 435 ctc cag gtt ttg tcc caa gta cct ttc cgt aagcta aat aaa aag aaa 1461 Leu Gln Val Leu Ser Gln Val Pro Phe Arg Lys LeuAsn Lys Lys Lys 440 445 450 gag aag tct aaa aag gaa aag aaa aaa gaa aaggtt aat aac agc aat 1509 Glu Lys Ser Lys Lys Glu Lys Lys Lys Glu Lys ValAsn Asn Ser Asn 455 460 465 gaa aat cca aga aaa atg tgt gag caa atg aggcta aag gaa aag tcc 1557 Glu Asn Pro Arg Lys Met Cys Glu Gln Met Arg LeuLys Glu Lys Ser 470 475 480 aag aga aat cag cca aag aaa agg aaa caa cagttc att ggt cta aaa 1605 Lys Arg Asn Gln Pro Lys Lys Arg Lys Gln Gln PheIle Gly Leu Lys 485 490 495 500 tct gaa gat gaa gat aat gct aaa cct atgaac tat gat gag aaa agg 1653 Ser Glu Asp Glu Asp Asn Ala Lys Pro Met AsnTyr Asp Glu Lys Arg 505 510 515 cag tta agt ctg aat ata aac aaa ctc cctgga gat aaa ctt ggg cga 1701 Gln Leu Ser Leu Asn Ile Asn Lys Leu Pro GlyAsp Lys Leu Gly Arg 520 525 530 gta gtt cac ata ata caa tca aga gag ccttct ctg agc aat tcc aat 1749 Val Val His Ile Ile Gln Ser Arg Glu Pro SerLeu Ser Asn Ser Asn 535 540 545 cct gat gag ata gag ata gac ttt gaa acactg aaa gca tca aca cta 1797 Pro Asp Glu Ile Glu Ile Asp Phe Glu Thr LeuLys Ala Ser Thr Leu 550 555 560 aga gaa tta gaa aaa tat gtt tcg gca tgtcta aga aag aga cca tta 1845 Arg Glu Leu Glu Lys Tyr Val Ser Ala Cys LeuArg Lys Arg Pro Leu 565 570 575 580 aaa cct cct gct aag aaa ata atg atgtcc aaa gaa gaa ctt cac tca 1893 Lys Pro Pro Ala Lys Lys Ile Met Met SerLys Glu Glu Leu His Ser 585 590 595 cag aaa aaa cag gaa ttg gaa aag cggtta ctg gat gtt aat aat cag 1941 Gln Lys Lys Gln Glu Leu Glu Lys Arg LeuLeu Asp Val Asn Asn Gln 600 605 610 tta aat tct aga aaa cgt caa aca aaatct gat aaa acg caa cca tcc 1989 Leu Asn Ser Arg Lys Arg Gln Thr Lys SerAsp Lys Thr Gln Pro Ser 615 620 625 aaa gct gtt gaa aat gtt tcc cga ctgagt gag agc agc agc agc agc 2037 Lys Ala Val Glu Asn Val Ser Arg Leu SerGlu Ser Ser Ser Ser Ser 630 635 640 agc agc tca tca gag tct gaa agt agcagc agt gac tta agc tct tca 2085 Ser Ser Ser Ser Glu Ser Glu Ser Ser SerSer Asp Leu Ser Ser Ser 645 650 655 660 gac agc agt gat tct gaa tca gaaatg ttc cct aag ttt aca gaa gta 2133 Asp Ser Ser Asp Ser Glu Ser Glu MetPhe Pro Lys Phe Thr Glu Val 665 670 675 aaa cca aat gat tct cct tct aaagag cat gta aag aaa atg aag aat 2181 Lys Pro Asn Asp Ser Pro Ser Lys GluHis Val Lys Lys Met Lys Asn 680 685 690 gaa tgc ata ctg cct gaa gga agaaca ggc gtc aca cag ata gga tat 2229 Glu Cys Ile Leu Pro Glu Gly Arg ThrGly Val Thr Gln Ile Gly Tyr 695 700 705 tgt gtg caa gac aca acc tct gccaat act acc ctt gtt cat cag acc 2277 Cys Val Gln Asp Thr Thr Ser Ala AsnThr Thr Leu Val His Gln Thr 710 715 720 aca cct tca cat gta atg cca ccaaat cac cac caa tta gca ttt aat 2325 Thr Pro Ser His Val Met Pro Pro AsnHis His Gln Leu Ala Phe Asn 725 730 735 740 tat caa gaa tta gaa cat ttacag act gtg aaa aac att tca cct tta 2373 Tyr Gln Glu Leu Glu His Leu GlnThr Val Lys Asn Ile Ser Pro Leu 745 750 755 caa att ctg cct ccc tca ggtgat tct gaa cag ctc tca aat ggc ata 2421 Gln Ile Leu Pro Pro Ser Gly AspSer Glu Gln Leu Ser Asn Gly Ile 760 765 770 act gtg atg cat cca tct ggtgat agt gac aca acg atg tta gaa tct 2469 Thr Val Met His Pro Ser Gly AspSer Asp Thr Thr Met Leu Glu Ser 775 780 785 gaa tgt caa gct cct gta cagaag gat ata aag att aag aat gca gat 2517 Glu Cys Gln Ala Pro Val Gln LysAsp Ile Lys Ile Lys Asn Ala Asp 790 795 800 tca tgg aaa agt tta ggc aaacca gtg aaa cca tca ggt gta atg aaa 2565 Ser Trp Lys Ser Leu Gly Lys ProVal Lys Pro Ser Gly Val Met Lys 805 810 815 820 tcc tca gat gag ctc ttcaac caa ttt aga aaa gca gcc ata gaa aag 2613 Ser Ser Asp Glu Leu Phe AsnGln Phe Arg Lys Ala Ala Ile Glu Lys 825 830 835 gaa gta aaa gct cgg acacag gaa ctc ata cgg aag cat ttg gaa caa 2661 Glu Val Lys Ala Arg Thr GlnGlu Leu Ile Arg Lys His Leu Glu Gln 840 845 850 aat aca aag gaa cta aaagca tct caa gaa aat cag agg gat ctt ggg 2709 Asn Thr Lys Glu Leu Lys AlaSer Gln Glu Asn Gln Arg Asp Leu Gly 855 860 865 aat gga ttg act gta gaatct ttt tca aat aaa ata caa aac aag tgc 2757 Asn Gly Leu Thr Val Glu SerPhe Ser Asn Lys Ile Gln Asn Lys Cys 870 875 880 tct gga gaa gag cag aaagaa cat ccg cag tca tca gaa gct caa gat 2805 Ser Gly Glu Glu Gln Lys GluHis Pro Gln Ser Ser Glu Ala Gln Asp 885 890 895 900 aaa tcc aaa ctc tggctt ctc aaa gac cgt gat tta gcc agg ccg aaa 2853 Lys Ser Lys Leu Trp LeuLeu Lys Asp Arg Asp Leu Ala Arg Pro Lys 905 910 915 gaa caa gag agg aggagg aga gaa gcc atg gtg ggt acc att gat atg 2901 Glu Gln Glu Arg Arg ArgArg Glu Ala Met Val Gly Thr Ile Asp Met 920 925 930 acc ctt caa agt gacatt atg aca atg ttt gaa aac aac ttt gat 2946 Thr Leu Gln Ser Asp Ile MetThr Met Phe Glu Asn Asn Phe Asp 935 940 945 taaaactcag tttttaaattaaccatccac ttaaaatgaa tggtaaaaga tcaaaatgca 3006 tatggtaaaa tgattgctttcagataacaa gataccaatc ttatattgta ttttgactgc 3066 tctaaaatga ttaaacagttttcacttaca aaaaaaaa 3104 3 22 DNA Artificial Sequence syntheticallygenerated primer 3 aatgtctctg ccaagtcgac aa 22 4 22 DNA ArtificialSequence synthetically generated primer 4 agcatccaca ggacgttgaa ag 22 515 PRT Homo sapiens 5 Cys Ser Glu Ile Leu Lys Glu Met Leu Ala Lys LysHis Phe Ser 1 5 10 15 6 11 PRT Homo sapiens 6 Tyr Tyr Thr Ile Ile LysAsn Pro Met Asp Leu 1 5 10 7 16 PRT Homo sapiens 7 Tyr Asn Lys Pro GlyAsp Asp Ile Val Leu Met Ala Gln Ala Leu Glu 1 5 10 15 8 15 PRT Homosapiens 8 Cys Ser Glu Ile Leu Lys Glu Met Leu Ala Lys Lys His Phe Ser 15 10 15 9 10 PRT Homo sapiens 9 Tyr Asp Val Val Lys Asn Arg Met Asp Leu1 5 10 10 7 PRT Homo sapiens 10 Tyr Lys Asp Ala Tyr Ser Phe 1 5 11 17PRT Homo sapiens 11 Tyr Asn Pro Pro Asp His Glu Val Val Thr Met Ala ArgMet Leu Gln 1 5 10 15 Asp 12 8 PRT Homo sapiens 12 Asn Tyr Asp Glu LysArg Gln Leu 1 5 13 15 PRT Homo sapiens 13 Tyr Leu His Lys Val Val MetLys Ala Leu Trp Lys His Gln Phe 1 5 10 15 14 11 PRT Homo sapiens 14 TyrHis Lys Ile Ile Lys Gln Pro Met Asp Met 1 5 10 15 16 PRT Homo sapiens 15Tyr Asn Lys Pro Thr Asp Asp Ile Val Leu Met Ala Gln Thr Leu Glu 1 5 1015 16 15 PRT Homo sapiens 16 Cys Asn Gly Ile Leu Lys Glu Leu Leu Ser LysLys His Ala Ala 1 5 10 15 17 10 PRT Homo sapiens 17 Tyr His Asp Ile LysHis Pro Met Asp Leu 1 5 10 18 7 PRT Homo sapiens 18 Tyr Arg Asp Ala GlnGlu Phe 1 5 19 17 PRT Homo sapiens 19 Tyr Asn Pro Pro Asp His Asp ValVal Ala Met Ala Arg Lys Leu Gln 1 5 10 15 Asp 20 8 PRT Homo sapiens 20Ser Tyr Asp Glu Lys Arg Gln Leu 1 5 21 15 PRT Drosophila melanogaster 21Tyr Ile Leu Lys Thr Val Met Lys Val Ile Trp Lys His His Phe 1 5 10 15 2211 PRT Drosophila melanogaster 22 Tyr His Lys Ile Ile Lys Gln Pro MetAsp Met 1 5 10 23 16 PRT Drosophila melanogaster 23 Tyr Asn Lys Pro GlyGlu Asp Val Val Val Met Ala Gln Thr Leu Glu 1 5 10 15 24 15 PRTDrosophila melanogaster 24 Cys Asn Glu Ile Leu Lys Glu Leu Phe Ser LysLys His Ser Gly 1 5 10 15 25 10 PRT Drosophila melanogaster 25 Tyr HisAsp Ile Lys His Pro Met Asp Leu 1 5 10 26 7 PRT Drosophila melanogaster26 Tyr Gln Ser Ala Pro Glu Phe 1 5 27 17 PRT Drosophila melanogaster 27Tyr Asn Pro Pro Asp His Asp Val Val Ala Met Gly Arg Lys Leu Gln 1 5 1015 Asp 28 8 PRT Drosophila melanogaster 28 Ser Tyr Asp Glu Lys Arg GlnLeu 1 5

What is claimed is:
 1. A substantially pure polypeptide comprising anamino acid sequence at least 60% identical to SEQ ID NO:1, wherein thepolypeptide regulates transcription of a gene and comprises abromodomain.
 2. The polypeptide of claim 1, wherein the amino acidsequence is at least 70% identical to SEQ ID NO:1.
 3. The polypeptide ofclaim 1, wherein the amino acid sequence is at least 80% identical toSEQ ID NO:1.
 4. The polypeptide of claim 1, wherein the amino acidsequence is at least 90% identical to SEQ ID NO:1.
 5. A substantiallypure polypeptide comprising SEQ ID NO:1.
 6. A substantially purepolypeptide comprising the amino acid sequence of SEQ ID NO:1, with upto 30 conservative amino acid substitutions, wherein the polypeptideregulates transcription of a gene and comprises a bromodomain.
 7. Asubstantially pure polypeptide encoded by a nucleic acid that hybridizesunder high stringency conditions to a probe the sequence of whichconsists of SEQ ID NO:2, wherein the polypeptide regulates transcriptionof a gene and comprises a bromodomain.
 8. An isolated nucleic acidencoding the polypeptide of claim
 1. 9. An isolated nucleic acidencoding the polypeptide of claim
 5. 10. An isolated nucleic acidencoding the polypeptide of claim
 6. 11. An isolated nucleic acidcomprising a strand that hybridizes under high stringency conditions toa single stranded probe consisting of SEQ ID NO:2.
 12. The isolatednucleic acid of claim 11, wherein the nucleic acid encodes a polypeptidethat regulates transcription of a gene and comprises a bromodomain. 13.The nucleic acid of claim 12, wherein the polypeptide comprises SEQ IDNO:1.
 14. The nucleic acid of claim 11, wherein the strand is at least15 nucleotides in length.
 15. A vector comprising the nucleic acid ofclaim
 8. 16. A vector comprising the nucleic acid of claim
 9. 17. Avector comprising the nucleic acid of claim
 10. 18. A vector comprisingthe nucleic acid of claim
 11. 19. A vector comprising the nucleic acidof claim
 12. 20. A cultured host cell comprising the nucleic acid ofclaim
 8. 21. A cultured host cell comprising the nucleic acid of claim9.
 22. A cultured host cell comprising the nucleic acid of claim
 10. 23.A cultured host cell comprising the nucleic acid of claim
 11. 24. Acultured host cell comprising the nucleic acid of claim
 12. 25. Anantibody that specifically binds to the polypeptide of claim
 1. 26. Amethod of preparing a polypeptide, the method comprising culturing thehost cell of claim 20, wherein the host cell expresses the polypeptide,and isolating the polypeptide from the host cell.
 27. A method ofscreening for a compound that binds to a polypeptide, the methodcomprising providing a polypeptide comprising an amino acid sequence atleast 60% identical to SEQ ID NO:1; contacting a test compound with thepolypeptide; and determining whether the test compound has bound to thepolypeptide.
 28. A compound which specifically binds to the polypeptideof claim 1.